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REVIEW
Division of Molecular Biology of the Cell II, German Cancer Research Center, D-69120 Heidelberg, Germany
Mammalian cells contain 100 or more copies of tandemly repeated ribosomal
RNA (rRNA) genes per haploid genome. These genes are transcribed with high
efficiency to keep up with the cell's metabolic activity and demand for
ribosomes. Alterations in cell proliferation are accompanied by profound
changes in the transcription rate of rRNA genes. Thus, by responding to
changes in the cellular environment, transcription by RNA polymerase I (Pol I)
ultimately determines ribosome production and the potential for cell growth
and proliferation. There are several comprehensive reviews that discuss
regulation of rRNA synthesis in vertebrates and yeast
(Grummt 1999
;
Reeder 1999;
Warner 1999;
Moss and Stefanovsky 2002).
However, new data have been produced even since the latest of these reviews
that uncover the mechanisms that link Pol I transcription to cellular
physiology. In this review, I restrict the background information to the
minimal level that is required for understanding initiation complex formation
at the rDNA promoter before proceeding to review the regulatory pathways that
adapt cellular rRNA synthesis to cell metabolism and growth.
 |
Structural organization of the rRNA transcription unit |
|---|
). With a few exceptions, rDNA promoters share a common
modular organization, consisting of a start site proximal core promoter (CP)
and an upstream control element (UCE). The stereospecific alignment of both
sequence elements is crucial for efficient transcription initiation. Analysis
of structural parameters of ribosomal gene promoters from human to lower
plants revealed the conservation of specific structural features, rather than
base sequence, that are fundamental for promoter function
(Marilley and Pasero 1996;
Marilley et al. 2002).
Apparently, a structural code, in addition to primary sequence, directs
specific DNA–protein interactions at the rDNA promoter and may play an
important function in transcriptional control. |
Basal Pol I transcription initiation factors |
|---|
) and the promoter selectivity factor, termed TIF-IB in
mouse (Clos et al. 1986) and
SL1 in humans (Learned et al.
1985). UBF contains several HMG boxes, a motif known to bend DNA.
Like other HMG proteins, UBF interacts with the minor groove of DNA and binds
to structured nucleic acids such as kinked DNA, cruciforms, or four-way
junctions (Putnam et al.
1994). The tandem HMG boxes enable a single dimer of UBF to wrap
the DNA in a right-handed direction, forming a loop of almost 360° once
every 140 bp, thereby bringing the core and the UCE into close proximity
(Bazett-Jones et al. 1994;
Copenhaver et al. 1994). This
structure may provide the correct scaffolding for productive interactions
between UBF and TIF-IB/SL1 bound to the two promoter elements and facilitate
initiation complex formation. UBF is known to activate rDNA transcription by
recruiting Pol I to the rDNA promoter, stabilizing binding of TIF-IB/SL1, and
competing with nonspecific DNA-binding proteins, such as histone H1
(Kuhn and Grummt 1992;
Kuhn et al. 1993). Moreover,
UBF has been shown to bind to nucleosomes and displace histone H1 from the
linker region on preassembled nucleosomes
(Kermekchiev et al. 1997).
|
Promoter specificity is conferred by TIF-IB/SL1, a protein complex
containing the TATA-binding protein (TBP) and three Pol I-specific
TBP-associated factors TAFI48, TAFI68,
TAFI95/110 (Comai et al.
1992; Eberhard et al.
1993; Heix et al.
1997). In contrast to TFIID, the factor that nucleates Pol II
transcription initiation complexes, the TBP subunit of TIF-IB/SL1 does not
bind to DNA, and promoter recognition is carried out by the associated
TAFIs. As is discussed below, the most important step in the
assembly of a productive transcription initiation complex is the recruitment
of Pol I to the rDNA promoter. This is achieved by interaction of UBF with
PAF53, the mammalian homolog of the yeast Pol I subunit A49
(Hanada et al. 1996;
Seither et al. 1997), and by
interaction of TIF-IB/SL1 with TIF-IA. TIF-IA is the mammalian homolog of
yeast Rrn3p (Bodem et al. 2000;
Moorefield et al. 2000), a
regulatory factor that is associated with the initiation-competent
subpopulation of Pol I (Miller et al.
2001; Yuan et al.
2002).
In Saccharomyces cerevisiae, rDNA transcription requires Pol I,
the TATA-binding protein (TBP), Rrn3p, the core factor (CF), and the upstream
activating factor (UAF). CF is composed of three stably associated proteins
encoded by RRN6, RRN7, and RRN11. UAF is a complex of six
polypeptides including Rrn5p, Rrn9p, Rrn10p, the two histones H3 and H4, and
Uaf30p (see Fig. 2; for review,
see Nomura 2001). Binding of
UAF to the promoter is necessary to recruit CF and the Pol I–Rrn3p
complex. Transcription experiments with immobilized templates revealed that CF
and TBP, along with Pol I and Rrn3p, are released from the template upon
transcription, whereas UAF remains associated with the upstream promoter
element, presumably serving as a scaffold for reinitiation
(Aprikian et al. 2001). These
findings support a model in which essential components of the Pol I machinery
cycle on and off the promoter with each round of transcription. In contrast,
similar experiments with human Pol I and the respective factors demonstrated
recycling of Pol I and TIF-IA/Rrn3p, but not of UBF and TIF-IB/SL1
(Panov et al. 2001).
|
|
The RNA polymerase I holoenzyme concept |
|---|
; Seither et al.
1998; Albert et al.
1999; Hannan et al.
1999). Although the composition of these large protein complexes
is not well defined, they all contain Pol I and basal transcription factors to
support rDNA transcription in vitro and additional polypeptides that play a
role in protein phosphorylation (CKII), chromatin modification (PCAF), or DNA
repair/replication (topoisomerase I, Ku70/80, and PCNA). Interestingly, TFIIH,
the protein complex that plays an essential role in transcription of
protein-coding genes and nucleotide excision repair, was also found to be an
integral part of the Pol I holoenzyme
(Iben et al. 2002). These
findings are compatible with a mechanism by which Pol I is recruited to the
rDNA promoter as a giant multiprotein complex that contains proteins required
for rRNA synthesis and maturation, chromatin modification, and DNA repair. In
support of this view, a novel ribonucleoprotein complex enriched in nucleolar
proteins has been purified from yeast that contained Pol I, TBP, Rrn3p, Rrn5p,
Rrn7p, and Reb1p along with rRNA-processing factors like Nop1p, Cbf5p, Nhp2p,
and Rrp5p as well as small nucleolar RNAs
(Fath et al. 2000). The
ribonucleoprotein complex supports accurate transcription, termination, and
pseudouridylation of rRNA, suggesting that it represents a nucleolar subdomain
that serves as a scaffold for coordinated rRNA synthesis and processing.
However, it cannot be excluded that after breakage of the cells, such
complexes are artificially generated by specific or non-specific
macromolecular interactions. In support of this, recent FRAP experiments have
demonstrated that Pol I subunits enter the nucleolus as distinct subunits
rather than as a preassembled complex. These observations suggest that
assembly of Pol I and functional initiation complexes may proceed in a
sequential manner via metastable intermediates, each with increasing stability
as more subunits are added (Dundr et al.
2002). |
Dynamics of the Pol I transcription apparatus |
|---|
). Using
green fluorescent protein (GFP) tags that permit the observation of proteins
in living cells by the FRAP (fluorescent recovery after photobleaching)
technique, the kinetics of assembly and elongation of mammalian Pol I has been
analyzed. The data revealed that the Pol I transcription machinery is a highly
dynamic complex that assembles in a stochastic fashion from freely diffusible
subunits. Each of the components is steadily and rapidly exchanged between the
nucleoplasm and the nucleolus. Real-time evaluation of promoter-associated
multiprotein complexes revealed occupancy of only a few seconds, indicating
that the association of transcription factors with their binding sites is
transient. A hit-and-run mechanism was proposed, in which transcriptional
factors quickly exchange between individual rDNA promoters. Pol I subunits
have been found to enter the nucleolus as distinct subunits rather than as a
preassembled holoenzyme. Surprisingly, Pol I appears to break apart after
transcription termination and needs to reassemble before transcription
initiation. Calculations of the FRAP data indicate that transcription
initiation at a ribosomal promoter occurs every 
1.4 sec, Pol I subunits
reside in the pool for 9 to 37 sec, and the residence time of
elongating Pol I is 2–3 min. With the use of computational modeling of
imaging data, the in vivo elongation time of Pol I has been determined as
140 sec, corresponding to an elongation rate of 95 nt/sec for a human
rDNA gene of 13.3 kb. Although one can question whether imaging and
mathematical models can provide such an unambiguous picture of assembly, the
estimated numbers are approximately the same as those obtained by French et
al. (2003), who calculated the
elongation rate of yeast Pol I directly from the number of Pol I molecules per
rRNA gene and the rate of rRNA synthesis. |
Mechanisms regulating Pol I transcription |
|---|
). Changes in Pol I
transcription regulate ribosome production and thus determine the potential
for cell proliferation. rDNA genes are present in multiple copies, and
therefore, rRNA synthesis could be modulated by varying the transcription rate
per gene or by varying the number of active genes. Although there are several
reports demonstrating that in yeast both of these mechanisms may operate under
certain conditions (for review, see Reeder
1999), a recent electron microscopy study revealed that the
overall initiation rate, and not the number of active genes, determines the
rate of rDNA transcription during exponential growth in yeast
(French et al. 2003).
Similarly, in vertebrates the level of cellular rRNA is regulated by changing
the rate of transcription initiation at active rDNA genes rather than by
activating silent transcription units. In vivo psoralen-cross-linking studies
that can distinguish between transcriptionally active and inactive genes have
revealed that even in exponentially growing mammalian cells that synthesize
high levels of pre-rRNA, only half of the rDNA genes are transcriptionally
active and maintained in an "open" chromatin conformation. The
other half that corresponds to inactive gene copies resides in a compact,
nucleosomal structure. The ratio of active and inactive rRNA genes is stably
propagated through the cell cycle and is independent of the cellular rRNA
synthetic activity (Conconi et al.
1989). The present view is that growth-dependent modulation of Pol
I transcription occurs at transcriptionally competent gene copies, and
"opening" or "closing" of ribosomal genes is not
involved in short-term Pol I transcription regulation. Epigenetic mechanisms
that mediate rDNA silencing will be discussed in another review
(Grummt and Pikaard 2003).
Early studies in mice and rats have demonstrated up or down-regulation of
rRNA synthesis after partial hepatectomy, hormone administration,
cycloheximide treatment, or nutrient starvation (for reviews, see
Jacob 1995;
Grummt 1999). Deprivation of a
single amino acid from the culture medium has been demonstrated to cause a
rapid shut-off of nucleolar transcription
(Grummt et al. 1976).
Meanwhile, several studies have been published that address the mechanisms
underlying cell-cycle- and growth-factor-dependent fluctuations of Pol I
activity. Indeed, almost any perturbation that slows down cell growth or
protein synthesis decreases rDNA transcription. Evidence accumulated to date
indicates that almost any of the proteins required for Pol I transcription can
serve as a target for regulatory pathways. For example, changes in the
phosphorylation pattern of UBF play a key role in modulating rDNA activity
during cell cycle progression. UBF is phosphorylated at multiple sites, and
phosphorylation of the C terminus by casein kinase II facilitates the
interaction between UBF and TIF-IB/SL1
(Tuan et al. 1999). In
quiescent cells, UBF is hypophosphorylated and transcriptionally inactive
(O'Mahony et al. 1992; Voit et
al. 1992,
1995). Moreover, interactions
with pRb, p130, and p53 have been shown to impair UBF functions, such as DNA
binding or the interaction with TIF-IB/SL1
(Cavanaugh et al. 1995;
Voit et al. 1997;
Budde and Grummt 1999;
Zhai and Comai 2000;
Ciarmatori et al. 2001).
Finally, acetylation of UBF by the histone acetyltransferase CBP has been
reported to enhance UBF activity in vitro, and overexpression of both CBP and
p300 enhances Pol I activity in vivo (Hirschler-Lankiewicz et al. 2001).
Besides UBF, the TAFI68 subunit of TIF-IB/SL1 is acetylated by
PCAF. Acetylation enhances binding of TAFI68 to rDNA and augments
Pol I transcription. Conversely, deacetylation of TAFI68 by the
NAD+-dependent histone deacetylase mSir2a represses Pol I
transcription (Muth et al.
2001). As is discussed below, there is evidence for changes in the
phosphorylation pattern of SL1 (Heix et
al. 1998), UBF (Klein and
Grummt 1999; Voit et al.
1999), and TTF-I (Sirri et al.
2000,
2002) that correlate with
cell-cycle-specific fluctuations of rDNA transcription. Thus, reversible
acetylation and phosphorylation of basal components of the Pol I transcription
machinery may be an effective means to regulate rDNA transcription.
|
Growth-dependent transcription regulation by TIF-IA |
|---|
;
Schnapp et al. 1993;
Bodem et al. 2000;
Moorefield et al. 2000). A
preinitiation complex containing Pol I can be formed in the absence of
TIF-IA/Rrn3p; however, formation of the first phosphodiester bond requires the
presence of TIF-IA/Rrn3p (Schnapp and
Grummt 1991; Schnapp et al.
1993). Following initiation, TIF-IA is released from the ternary
complex and can associate with another preinitiation complex. The activity of
TIF-IA/Rrn3p is regulated by diverse extracellular signals, suggesting that
this factor adapts Pol I transcription to cell growth. In both mammals and
yeast, a large fraction of Pol I, the "bulk" enzyme, termed Pol
I
, is unable to support specific initiation, despite its ability to
synthesize RNA from nonspecific templates
(Tower and Sollner-Webb 1987;
Schnapp et al. 1990;
Miller et al. 2001). Only the
fraction of Pol I (Pol I
) that is associated with TIF-IA/Rrn3p is
capable of assembling into a productive initiation complex
(Yamamoto et al. 1996;
Milkereit and Tschochner
1998), suggesting that TIF-IA/Rrn3p bridges Pol I to the
preinitiation complex. Importantly, the amount of TIF-IA/Rrn3p associated with
Pol I, but not the overall level of TIF-IA/Rrn3p, is decreased in
growth-arrested cells, indicating that transcriptional shut-off is caused by
dissociation of the Pol I/Rrn3p complex
(Milkereit and Tschochner
1998; Cavanaugh et al.
2002; Yuan et al.
2002). These and other experiments demonstrate that most, if not
all, growth-dependent control of rDNA transcription may be exerted by the
formation and recruitment of TIF-IA/Rrn3p–Pol I complexes to the rDNA
promoter. This suggests a regulatory cycle in which TIFIA/Rrn3p dissociates
from Pol I during initiation or after promoter escape, is inactivated after
release, and must be reactivated before association with another polymerase
and assembly into a new preinitiation complex
(Aprikian et al. 2001).
The role of TIF-IA/Rrn3p as a bridge between Pol I and TIF-IB/SL1 or CF,
respectively, has been supported by genetic and biochemical experiments in
S. cerevisiae and mammals demonstrating that TIF-IA/Rrn3p interacts
with RPA43, a unique subunit of Pol I
(Peyroche et al. 2000;
Fath et al. 2001;
Cavanaugh et al. 2002;
Yuan et al. 2002). In
addition, TIF-IA has been found to interact with PAF67, a 67-kD Pol
I-associated factor that decorates the initiation-competent form of Pol I
(Seither et al. 2001). This
suggests that by interacting with PAF67, TIF-IA may target a functional subset
of Pol I molecules into a productive transcription initiation complex. TIF-IA
also interacts with two TAFI subunits of TIF-IB/SL1
(Miller et al. 2001;
Yuan et al. 2002) and the
Rrn6p subunit of CF (Peyroche et al.
2000). Thus, by associating with both Pol I and the promoter
selectivity factor, TIF-IA may link both protein complexes.
Given the essential role for TIF-IA/Rrn3p in targeting Pol I to
promoter-bound TIF-IB/SL1, the interactions with TAFIs, RPA43, and
PAF67 are expected to be major targets of regulatory pathways that control the
assembly of Pol I preinitiation complexes. Indeed, interactions between
TIF-IA/Rrn3p with Pol I are affected by diverse regulatory pathways that link
the cell's biosynthetic activities to environmental conditions. Nutrient
starvation, density arrest, and protein synthesis inhibitors lead to
inactivation of TIF-IA (Cavanaugh et al.
2002; Yuan et al.
2002). TIF-IA is phosphorylated at multiple sites, and signals
that affect cell metabolism alter the phosphorylation pattern of TIF-IA
(Zhao et al. 2003). In
density-arrested, cycloheximide-treated, and amino-acid-starved cells, TIF-IA
is hypophosphorylated and incapable of binding to Pol I
(Yuan et al. 2002). Thus,
cellular signaling cascades directly target TIF-IA, and reversible
phosphorylation regulates the association of TIF-IA with Pol I and hence
transcription initiation complex formation.
|
TOR and MAP kinase signaling pathways target TIF-IA |
|---|
). The
TOR/p70S6k pathway controls translation, ribosome biogenesis, and
many growth-related processes in response to nutrients and environmental
conditions. Nutrient deprivation inhibits mTOR kinase activity and blocks cell
growth. The mechanism by which TOR senses nutrient availability is unknown, as
is its involvement in regulation of rDNA transcription. Extracts from cells
treated with the mTOR inhibitor rapamycin are transcriptionally inactive
(Mahajan 1994;
Zaragoza et al. 1998), and
treatment of yeast cells with rapamycin leads to inhibition of rRNA synthesis
(Powers and Walter 1999).
Interestingly, transcriptional activity of rapamycin-treated cell extracts can
be restored by mTOR, p70S6k, or recombinant TIF-IA (I. Grummt,
unpubl.). This suggests that phosphorylation by mTOR, p70S6k, or
downstream kinase(s) is required for TIF-IA activity. In support of this, the
assembly of TIF-IA/Pol I complexes has been found to be impaired in
rapamycin-treated cells. Thus, TOR/p70S6k signaling regulates rDNA
transcription by modulating the activity of TIF-IA.
A different pathway, but similar scenario, mediates transcriptional
activation by growth factors. After mitogenic stimulation of quiescent cells,
a transient 10-fold increase in pre-rRNA synthesis was observed
(Zhao et al. 2003). The rapid
response of rDNA transcription to growth factors was blocked by PD98059, an
inhibitor of MEK1/2, indicating that Pol I transcription is up-regulated by
activation of the Ras–ERK pathway. Transcriptional activation correlates
with phosphorylation of TIFIA at two specific serine residues (S633 and S649)
by ERK and RSK kinases. Phosphorylation at these serine residues activates
TIF-IA and increases cellular pre-rRNA synthesis. Replacement of Ser 649 by
alanine, on the other hand, abolishes TIF-IA activity, impairs Pol I
transcription in vivo and in vitro, and retards cell growth. Thus, growth
factors regulate rRNA synthesis and nucleolar activity by ERK/RSK-mediated
phosphorylation of TIF-IA (Fig.
3). These results underscore the molecular cross-talk between the
p70S6k and ERK signaling pathways
(Wang et al. 2001) and
demonstrate that TIF-IA is a common final target for growth factor-dependent
activation of ribosome biogenesis.
|
One additional point is worth mentioning. TIF-IA contains a conserved
sequence motif, known as the Walker type A or P-loop motif. The P-loop is a
flexible glycine-rich sequence that is embedded in a well-defined tertiary
structure and has been implicated in ATP and GTP binding
(Walker et al. 1982). The
presence of a potential ATP- and GTP-binding site in TIF-IA is interesting,
because rRNA synthesis in mouse cells has been shown to be regulated by the
intracellular pool sizes of ATP and GTP
(Grummt and Grummt 1976).
Moreover, recent studies in Escherichia coli have demonstrated that
the concentration of initiating nucleoside triphosphates, that is, ATP or GTP,
regulate rRNA transcription in a growth-rate-dependent manner
(Gaal et al. 1997). This
suggests that NTP-sensing by rrn P1 promoters links cellular rRNA
synthesis to the level of translation and the available energy resources
(Schneider et al. 2002). It is
tempting to speculate that eukaryotes use a similar ATP/GTP-sensing mechanism
to integrate extracellular signals into growth-rate-dependent regulation of
rRNA synthesis. Alternatively, the pool sizes of ATP and GTP could regulate
TIF-IA activity indirectly as a consequence of an effect on mTOR signaling.
mTOR itself has been shown to function as an ATP sensor, and mTOR signaling is
controlled by intracellular ATP concentrations
(Dennis et al. 2001). This
finding, together with the role of mTOR signaling in the regulation of TIF-IA
activity, may provide a link between nutrient availability, cellular ATP
levels, and regulation of rRNA synthesis.
|
TFIIH and CSBlink Pol I transcription to DNA repair |
|---|
).
The most striking feature of TFIIH is its multifunctionality. TFIIH is
engaged in promoter opening and phosphorylation of the C-terminal domain of
Pol II in the context of mRNA transcription and DNA opening in the setting of
nucleotide excision and transcription-coupled repair. In addition, TFIIH
serves an essential role in Pol I transcription. GFP-tagged TFIIH is
homogenously distributed through the nucleoplasm, with disperse clusters
colocalizing with Pol I in nucleoli
(Hoogstraten et al. 2002).
Electron microscopy and immunogold labeling have shown enrichment of TFIIH at
the dense fibrillar component of nucleoli, that is, sites of active rDNA
transcription (Iben et al.
2002). Microinjection of antibodies against subunits of TFIIH
induced a strong, rapid reduction of rRNA synthesis, demonstrating the
requirement of TFIIH in rDNA transcription. In yeast strains carrying
temperature-sensitive mutations in Tfb1 and Kin28, the homologs of mammalian
p62 and Cdk7, pre-rRNA synthesis declines at a similar rate as in Pol I
mutants upon shift to the restrictive temperature. Moreover, biochemical
studies have revealed that TFIIH is associated with a subpopulation of
TIF-IB/SL1 as well as with the initiation-competent form of Pol I (Pol
I). Reconstituted transcription systems lacking TFIIH are
transcriptionally inactive, and transcriptional activity can be restored by
purified TFIIH (Iben et al.
2002). TFIIH is required for productive but not abortive rDNA
transcription, implying a role in transcription elongation. These findings
suggest that errors in the DNA template encountered during transcription might
be corrected by TFIIH-mediated processes. Noteworthy, recent in vivo
photobleaching studies have revealed that TFIIH moves freely and is capable of
rapid switching between Pol I, Pol II, and NER complexes with an average
residence time of 25 sec, 6 sec, and 4 min, respectively
(Hoogstraten et al. 2002).
Thus, a stochastic exchange of TFIIH occurs between different multiprotein
complexes involved in different DNA transactions.
The close interrelationship between DNA repair and rRNA synthesis has
further been documented by the finding that CSB, a protein that is defective
in Cockayne's syndrome (CS), is required for Pol I transcription. CSB is
localized at sites of rDNA transcription and restores rRNA synthesis when
transfected in CSB-deficient cells. CSB is part of a megadalton-size protein
complex that contains Pol I, TFIIH, and basal Pol I transcription initiation
factors and promotes efficient rRNA synthesis in vitro
(Bradsher et al. 2002).
Mutations in CSB, as well as XPB and XPD genes, all
of which confer Cockayne's syndrome, disturb the Pol I/TFIIH/CSB complex and
reduce rRNA synthesis in vivo. The fragility of this complex could be the
molecular basis for some of the clinical features that are associated with the
CS phenotype.
|
Regulation of Pol I transcription during the cell cycle |
|---|
). In mammalian cells, on the other hand, rDNA transcription
strongly oscillates during cell cycle progression. Transcription is maximal in
the S and G2 phases, shuts down in mitosis, and slowly recovers in G1. Mitotic
silencing of human Pol I transcription is caused by phosphorylation of the
TAFI110 subunit of SL1 by cdk1/cyclin B at Thr 852
(Heix et al. 1998;
Kuhn et al. 1998). As a
consequence of this specific phosphorylation, the capability of TIF-IB/SL1 to
interact with UBF is impaired, and Pol I transcription is repressed. Moreover,
UBF is inactivated during mitosis, presumably both by loss of essential
phosphorylations and mitosis-specific inhibitory phosphorylation(s) (I.
Grummt, unpubl.). Thus, reversible phosphorylation of TIF-IB/SL1 and UBF is
used as a molecular switch to shut down rDNA transcription during mitosis. The
mechanisms that trigger reactivation of transcription at the end of mitosis
are unknown. Conceivably, cellular phosphatases have to reverse cdk1/cyclin
B-mediated phosphorylations to recover TIF-IB/SL1 activity during telophase. A
candidate enzyme for reactivation of the Pol I transcription machinery could
be Cdc14B, a phosphatase that is sequestered in an inactive state in the
nucleolus for most of the cell cycle and is released into the nucleus and
cytoplasm at the exit from mitosis
(Mailand et al. 2002).
In early G1 phase, rDNA transcription remains low although the activity of
TIF-IB/SL1 has been fully restored. The key player for activation of Pol I
transcription during G1 progression is UBF. To achieve high levels of rRNA
synthetic activity, UBF has to be phosphorylated at two serine residues by
G1-specific protein kinases. Cdk4/cyclin D1 targets Ser 484
(Voit et al. 1999), and cdk
2/cyclin E&A phosphorylates Ser 388
(Voit and Grummt 2001).
Mutations that prevent phosphorylation of Ser 388 impair the ability of UBF to
associate with Pol I and abrogate transcription. The finding that specific
cdk/cyclin complexes modulate the activity of TIF-IB/SL1 and UBF in a
cell-cycle-dependent manner links the control of cell cycle progression to
regulation of Pol I transcription (Fig.
4).
|
Another link between cell cycle regulation and rDNA transcription has
recently been uncovered by the finding that TAF1, the largest subunit of the
Pol II-specific TFIID complex, binds to UBF
(Lin et al. 2002). TAF1 (also
known as CCG1) has been implicated in the regulation of G1-to-S-phase
progression (Hisatake et al.
1993; Ruppert et al.
1993). Interaction of TAF1 with UBF stimulates human rDNA
transcription in vivo and in vitro. The results suggest that TAF1 may be
specifically engaged in the regulation of genes, including the ribosomal
genes, that play a critical role in the coordinate control of cell growth and
division.
|
The nucleolar RENT complex |
|---|
).
Transcription of RNA polymerase II genes integrated within the rDNA array is
repressed, and this repression is dependent on both UAF
(Vu et al. 1999) and Sir2
(silent information regulator #2), a protein that is conserved from archaea to
metazoa. The repressive chromatin structure associated with rDNA silencing
also functions in suppressing recombination among rDNA repeats, increasing
rDNA stability and extending the yeast life span
(Gottlieb and Esposito 1989).
Mutations that inactivate Sir2 shorten the yeast life span, and overexpression
of Sir2 extends it (Kaeberlein et al.
1999). Sir2 has been shown to be an NAD+-dependent
histone deacetylase (Imai et al.
2000), implying that Sir2-induced transcriptional silencing is
brought about by deacetylation of either histones or components of the
transcription apparatus.
In budding yeast, Sir2 executes functions in rDNA transcription as a
component of a nucleolar complex designated RENT (regulator of
nucleolar silencing and telophase exit), consisting of at least
three proteins, Sir2, Net1, and Cdc14. Net1, the core subunit of the RENT
complex, localizes Sir2 to rDNA and is required for silencing (for review, see
Guarente 2000). Net1
physically interacts with Pol I in vitro and stimulates rRNA synthesis
(Shou et al. 2001). Net1 and
Sir2 cross-link throughout individual rDNA repeats, and recent data
demonstrate that the Net1/Sir2 complex spreads unidirectionally downstream of
an active rDNA transcription unit (Buck et
al. 2002). Silencing requires transcription by Pol I, and the
direction of spreading is controlled by the direction of Pol I transcription.
To reconcile these findings, a model has been proposed in which the
interaction of Net1 with Pol I recruits Sir2 to active rDNA repeats for
histone deacetylation, and the unidirectional spreading of RENT/Sir2 is
mediated by its association with Pol I.
Besides its role in rDNA silencing, Net1 regulates both the exit from
mitosis and the activity of Cdc14, the third component of RENT
(Shou et al. 1999). The Cdc14
phosphatase is sequestered in an inactive state in the nucleolus and is
released from the RENT complex at the end of anaphase. Thus, destabilization
of the RENT complex appears to be a critical step in provoking exit from
mitosis and triggering cell cycle progression. It remains to be investigated
whether higher eukaryotes use a complex similar to RENT that links nuclear
integrity, transcriptional silencing, and cell cycle control.
|
Repression of rDNA transcription by the tumor suppressor proteins pRb and p53 |
|---|
). The related
"pocket" proteins pRb, p107, and p130 restrict cellular
proliferation and have been implicated in cell cycle regulation. The view that
pRb restrains cell proliferation by inactivating factors that are needed for
the transcription of genes required for DNA synthesis and cell proliferation
was challenged by the discovery that pRb also represses Pol I transcription.
UBF is the target for pRb-induced repression of Pol I transcription. pRb
accumulates in the nucleoli of differentiated or cell-cycle-arrested cells and
has been shown to repress rDNA transcription in vitro and in vivo
(Cavanaugh et al. 1995;
Voit et al. 1997;
Hannan et al. 2000).
Transcriptional repression is brought about by interaction of the C-terminal
part of pRb with HMG boxes 1 and 2 of UBF. Thus, inactivation of UBF appears
to be a most effective way for pRb to shut down rRNA synthesis and inhibit
cell growth. The acetyltransferase CBP that activates Pol I transcription by
acetylating UBF competes with pRb for binding to UBF, suggesting that the
competitive recruitment of CBP and pRb regulates UBF acetylation and rDNA
transcription (Pelletier et al.
2000). Interestingly, rRNA synthesis was unaffected in
Rb-/- cells, whereas Pol I transcription was elevated in cells
lacking either all three pocket proteins or pRb and p130. This suggests
overlapping functions of the pRb family members in the regulation of rRNA
synthesis. Consistent with such functional redundancy, the pocket protein p130
shares with pRb the ability to interact with UBF and repress Pol I
transcription in vivo and in vitro
(Ciarmatori et al. 2001).
Similar to pRb and p130, the tumor suppressor p53 has also been shown to
repress Pol I transcription in vivo and in vitro. Wild-type, but not mutant,
p53 can suppress Pol I transcription in cotransfection experiments, and
p53-deficient cells display increased pre-rRNA levels
(Budde and Grummt 1999;
Zhai and Comai 2000). p53
interacts with two subunits of SL1, TBP and TAFI110, which, in
turn, impairs initiation complex formation. These results reveal a novel
mechanism by which the tumor suppressors pRb and p53 inhibit cell
proliferation, namely, by direct inhibition of cellular rRNA synthesis. Given
that many tumor cells harbor mutations that affect both pRb and p53, the
combined effect of both mutations may have an added impact on Pol I activity
and tumor progression.
|
Conclusions and perspectives |
|---|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
E-MAIL I.Grummt{at}dkfz-heidelberg.de; FAX 49-6221-423404.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1098503R.
|
References |
|---|
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Structural organization of the...
Basal Pol I transcription...
